Pathogenic Role of Human Rhinovirus Mono-Infection in Pediatric Lower Respiratory Tract Infection.

Background and objective Human rhinovirus (HRV) is one of the leading causes of pediatric respiratory tract infection with a prevalence rate of 30-50%, mostly affecting children below five years of age and causing a substantial amount of economic loss. In children, it can alone or as a co-infection, cause a wide range of symptoms from mild to life-threatening ones. With the above background, the current study was carried out to emphasize the role of HRV mono-infection in pediatric acute respiratory tract infections by correlating clinical and molecular laboratory findings. Methods This study was carried out in a tertiary care teaching hospital over a duration of four years (March 2019-October 2023). Children up to 14 years of age visiting the outpatient department or admitted to the ward with diagnoses of acute respiratory tract infections (ARTIs) were included. The clinical and laboratory data were retrieved and analyzed. A nasopharyngeal swab (NPS) or throat swab (TS) was collected and sent to the Microbiology laboratory maintaining the cold chain. Nucleic acid was extracted and subjected to multiplex real-time polymerase chain reaction (RT-PCR). Result Of the 245 samples tested for the respiratory viral pathogen, 52 samples tested positive for HRV, of which 27 had HRV mono-infection. The clinico-demographic details of these 27 patients were studied in detail. The majority of the cases (24/27; 88.8%) were less than five years of age. Fever and shortness of breath were the most consistent symptoms in all. Nineteen (19/27; 62.9%) HRV mono-infection cases had underlying co-morbidities, all requiring respiratory support. The HRV mono-infection cases either developed bronchiolitis, lower respiratory tract infection, or pneumonia. All mono-infection cases had cycle threshold value (Ct) < 25, while the Ct value of HRV was > 30 in co-infection with other viruses. Conclusion Mono-infection of HRV in under-five children with underlying comorbidities and a lesser Ct value indicates severe disease manifestation and should be dealt with more cautiously.

Molecular characterization of coxsackievirus A24 variants isolated from an outbreak of acute hemorrhagic conjunctivitis.

PURPOSE: Acute Hemorrhagic conjunctivitis (AHC) is associated with CVA24v. Recently there was a severe outbreak of conjunctivitis in months of July and August 2023 in India. This study emphasizes the identification of the distinct mutations in the CVA24v strains, which were isolated during the AHC outbreak and could have potentially played a role in the high transmission of AHC in India during the 2023 outbreak. METHODS: A total of 71 conjunctivitis patients aged 1-75 years comprising 47 males and 24 females who attended Ophthalmology department of a tertiary care hospital of easternIndia were studied.RNA was extracted from all conjunctival swab samples and converted into cDNA. Subsequently, the viral 5' UTR was amplified and the PCR positive samples were subjected to sequencing. The newly isolated viral 5' UTR sequences were aligned with other worldwide sequences using the Clustal W tool to conduct mutational analysis. A phylogenetic tree was built using the MEGA software for viral genotype identification. RESULTS: All of the current outbreak strains belonged to genotype IV of CVA24v. The present outbreak strains formed a distinct clade in the phylogenetic tree and were different from previously reported Indian strains. Two persistent mutations, specifically in domain IV (T213C) and domain V (C475T), were exclusively detected within the internal ribosome entry site (IRES) of the 5' UTR of the current strains causing the outbreak. These two alterations have previously been shown to impact the virulence of another enterovirus (CV B3), but they have not been described in CVA24v until now. CONCLUSION: Finding of the present study highlights the possibility and the significance of the aforementioned two mutations in enhancing the transmissibility of the newer CVA24v strains. Hence, these two distinct mutations should be investigated further for developing antiviral therapies to combat future AHC outbreaks associated with CVA24v.

Salicylic Acid Conjugate of Telmisartan Inhibits Chikungunya Virus Infection and Inflammation.

There is no approved antiviral for the management of the Chikungunya virus (CHIKV). To develop an antiviral drug that can manage both CHIKV and arthritis induced by it, an ester conjugate of telmisartan (TM) and salicylic acid (SA) was synthesized (DDABT1). It showed higher potency (IC50 of 14.53 µM) and a good selectivity index [(SI = CC50/IC50) > 33]. On post-treatment of DDABT1, CHIKV infection was inhibited significantly by reducing CPE, viral titer, viral RNA, and viral proteins. Further, the time of addition experiment revealed >95% inhibition up to 4hpi indicating its interference predominantly in the early stages of infection. However, the late stages were also affected. This conjugate of SA and TM was found to increase the antiviral efficacy, and this might be partly attributed to modulating angiotensin II (Ang II) receptor type 1 (AT1). However, DDABT1 might have other modes of action that need further investigation. In addition, the in vivo experiments showed an LD50 of 5000 mg/kg in rats and was found to be more effective than TM, SA, or their combination against acute, subacute, and chronic inflammation/arthritis in vivo. In conclusion, DDABT1 showed remarkable anti-CHIKV properties and the ability to reduce inflammation and arthritis, making it a very good potential drug candidate that needs further experimental validation.

Human Bocavirus infection in childhood acute respiratory infection: Is it an innocent bystander?

PURPOSE: Acute respiratory infection (ARI) is one of the major attributing factors of under-five mortality and morbidity all over the world. Viruses are the most common cause of ARI. Due to the availability of molecular techniques, new viruses are getting isolated from children with ARI. With the above background, the present study was conducted to enlighten on the pathogenic role of human bocavirus (HBoV) in children with ARI. METHODOLOGY: This retrospective study was conducted over a period of >3 years duration. The clinical and laboratory data of the patients with signs and symptoms of ARI were retrieved and analyzed. Clinical profiles and outcome of the patients detected of having HBoV mono or co-infections were further analyzed in details. RESULTS: A total of 237 respiratory samples were subjected to respiratory panel by fast track diagnosis (FTD) multiplex polymerase chain reaction (multiplex PCR), of which 10 samples (mono-infection â€‹= â€‹4) were detected with the presence of HBoV. The clinical details of 8 cases were studied in details (details of rest 2 cases were missing). All the children were less than 3 years of age, with different co-morbid conditions such as low birth weight (n â€‹= â€‹4), cholestatic jaundice (n â€‹= â€‹1), operated case of congenital diaphragmatic hernia (n â€‹= â€‹1), pancytopenia (n â€‹= â€‹1), and primary immune deficiency (n â€‹= â€‹1). Their clinical course did not improve following antibiotic administration, 2 succumbed to death while the rest 6 cases were discharged. CONCLUSION: The present study highlights the fact that HBoV may not be an innocent bystander in the childhood ARI. Larger studies employing appropriate diagnostic modalities are needed to emboss it as a true pathogen and not merely a bystander.

6-Shogaol Exhibits Anti-viral and Anti-inflammatory Activity in COVID-19-Associated Inflammation by Regulating NLRP3 Inflammasomes.

Recent global health concern motivated the exploration of natural medicinal plant resources as an alternative target for treating COVID-19 infection and associated inflammation. In the current study, a phytochemical, 6-shogaol [1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one; 6-SHO] was investigated as a potential anti-inflammatory and anti-COVID-19 agent. In virus release assay, 6-SHO efficiently (94.5%) inhibited SARS-CoV2 replication. When tested in the inflammasome activation model, 6-SHO displayed mechanistic action by regulating the expression of the inflammasome pathway molecules. In comparison to the existing drugs, remdesivir and hydroxy-chloroquine, 6-SHO was not only found to be as effective as the standard anti-viral drugs but also much superior and safe in terms of predicted physicochemical properties and clinical toxicity. Comparative molecular dynamics simulation demonstrated a stable interaction of 6-SHO with NLRP3 (the key inflammasome regulator) in the explicit water environment. Overall, this study provides important cues for further development of 6-SHO as potential anti-inflammatory and anti-viral therapeutic agents.

DNA Damage Response Signaling Is Crucial for Effective Chikungunya Virus Replication.

Viruses utilize a plethora of strategies to manipulate the host pathways and hijack host machineries for efficient replication. Several DNA and few RNA viruses are reported to interact with proteins involved in DNA damage responses (DDRs). As the DDR pathways have never been explored in alphaviruses, this investigation intended to understand the importance of the DDR pathways in chikungunya virus (CHIKV) infection in vitro, in vivo, and ex vivo models. The study revealed that CHIKV infection activated the Chk2 and Chk1 proteins associated with the DDR signaling pathways in Vero, RAW264.7, and C2C12 cells. The comet assay revealed an increase in DNA damage by 95%. Inhibition of both ATM-ATR kinases by the ATM/ATR kinase inhibitor (AAKi) showed a drastic reduction in the viral particle formation in vitro. Next, the treatment of CHIKV-infected C57BL/6 mice with this drug reduced the disease score substantially with a 93% decrease in the viral load. The same was observed in human peripheral blood mononuclear cell (hPBMC)-derived monocyte-macrophage populations. Additionally, silencing of Chk2 and Chk1 reduced viral progeny formation by 91.2% and 85.5%, respectively. Moreover, CHIKV-nsP2 was found to interact with Chk2 and Chk1 during CHIKV infection. Furthermore, CHIKV infection induced cell cycle arrest in G1 and G2 phases. In conclusion, this work demonstrated for the first time the mechanistic insights regarding the induction of the DDR pathways by CHIKV that might contribute to the designing of effective therapeutics for the control of this virus infection in the future. IMPORTANCE Being intracellular parasites, viruses require several host cell machineries for effectively replicating their genome, along with virus-encoded enzymes. One of the strategies involves hijacking of the DDR pathways. Several DNA and few RNA viruses interact with the cellular proteins involved in the DDR pathways; however, reports regarding the involvement of Chk2 and Chk1 in alphavirus infection are limited. This is the first study to report that modulation of DDR pathways is crucial for effective CHIKV infection. It also reveals an interaction of CHIKV-nsP2 with two crucial host factors, namely, Chk2 and Chk1, for efficient viral infection. Interestingly, CHIKV infection was found to cause DNA damage and arrest the cell cycle in G1 and G2 phases for efficient viral infection. This information might facilitate the development of effective therapeutics for controlling CHIKV infection in the future.

MBZM-N-IBT, a Novel Small Molecule, Restricts Chikungunya Virus Infection by Targeting nsP2 Protease Activity In Vitro, In Vivo, and Ex Vivo.

The increase in disease incidences and persistent Chikungunya virus (CHIKV)-induced arthritis have been a huge burden on public health globally. In the absence of specific antivirals or vaccines, it is essential to continue efforts to develop effective anti-CHIKV strategies. Our previous study showing the in vitro anti-CHIKV potential of a novel molecule 1-[(2-methylbenzimidazol-1-yl) methyl]-2-oxo-indolin-3-ylidene] amino] thiourea (MBZM-N-IBT) encouraged us to further validate its efficacy. Here, the effect of MBZM-N-IBT was evaluated in vitro in RAW 264.7 cells, in vivo in C57BL/6 mice, and ex vivo in human peripheral blood mononuclear cells (hPBMCs). The study demonstrated that CHIKV infection was efficiently abrogated in RAW 264.7 cells (IC50 = 22.34 µM) with significant inhibition in viral proteins. The inhibition was effective in the postentry step, and MBZM-N-IBT predominately interfered in the early stages of CHIKV life cycle. It was further supported when the protease activity of CHIKV-nsP2 was hindered by the compound. Moreover, it diminished the CHIKV-induced inflammatory responses in vitro through significant downregulation of all the major mitogen-activated protein kinases (MAPKs), NF-κB, cyclooxygenase (COX)-2, and cytokines. Furthermore, MBZM-N-IBT restricted CHIKV infection and inflammation in vivo, leading to reduced clinical scores and complete survival of C57BL/6 mice. Additionally, it has been noticed that the CHIKV infection was reduced remarkably in hPBMC-derived monocyte-macrophage populations ex vivo by the compound. In conclusion, it can be suggested that this novel compound MBZM-N-IBT has been demonstrated to be a potential anti-CHIKV molecule in vitro, in vivo, and ex vivo and fulfilled all the criteria to investigate further for successful treatment of CHIKV infection.

Plants extracts from Cameroon pharmacopeia strongly inhibit the Chikungunya virus infection by targeting entry and replication steps.

ETHNOPHARMACOLOGICAL RELEVANCE: Cameroon is one of the sub-Saharan African countries affected by Chikungunya virus (CHIKV). With the absence of approved treatment, this disease represents globally a major public health concern. Several plants are traditionally used in Cameroon for the treatment of virus induced fever and arthralgia. But to date there is no study that validate the efficacy of these plants for the treatment of Chikungunya virus infection. AIM OF THE STUDY: This study aims to explore the inhition effect, mechanism of action of plant extracts against Chikungunya virus. MATERIAL AND METHODS: An ethnobotanical survey conducted in some regions of Cameroon, led to the identification of nine medicinal plants used in traditional medicine for the healing of fever-related diseases and arthritis. Crude hydro-ethanolic extracts of each plant were prepared by maceration and their effects against CHIKV infection were investigated. CHIKV S27 strain was used to infection in Vero cell line. The antiviral activities were determined by plaque assay and/or RT-PCR targeting E1 envelope gene of CHIKV. Dose-response studies of the active plants were also determined by flow cytometry and Western blot. RESULTS: Four extracts, Entada africana Guill et Pers. (E4), Entandrophragma cylindricum Sprague (EI), Khaya grandifoliola C. D.C. Sapindales (E2) and Macaranga hurifolia Beille (E6) showed antiviral activity with the half-maximal inhibitory concentration of 8.29; 8.14; 12.81 and 26.89 µg/mL respectively. All extracts were nontoxic up to the concentration of 100 µg/µL. Entandrophragma cylindricum Sprague (EI), Khaya grandifoliola C. D.C. Sapindales (E2), and Entada africana Guill et Pers. (E4) showed strong inhibition on the entry step of viral infection. At the same time, only Entandrophragma cylindricum Sprague (EI) inhibited the viral titer significantly in replication and intercellular assembly steps. Four plant extracts namely Entandrophragma cylindricum Sprague (EI), Macaranga hurifolia Beille (E6), Phragmentera capitata (Sprengel) Balle (E12), and Detarium microcarpum (E13) were effective against egression step. CONCLUSIONS: Together, the results of this study showed anti-chikungunya activities of Entandrophragma cylindricum Sprague (EI) and Macaranga hurifolia Beille (E6), with therapeutics perspectives and can be promising sources of the development of anti-CHIKV molecule in future.

Telmisartan Restricts Chikungunya Virus Infection In Vitro and In Vivo through the AT1/PPAR-γ/MAPKs Pathways.

Chikungunya virus (CHIKV) has reemerged as a global public health threat. The inflammatory pathways of the renin-angiotensin system (RAS) and peroxisome proliferator-activated receptor-gamma (PPAR-γ) are usually involved in viral infections. Thus, telmisartan (TM), which is known to block the angiotensin 1 (AT1) receptor and activate PPAR-γ, was investigated for activity against CHIKV. The anti-CHIKV effect of TM was investigated in vitro (Vero cells, RAW 264.7 cells, and human peripheral blood mononuclear cells [hPBMCs]) and in vivo (C57BL/6 mice). TM was found to abrogate CHIKV infection efficiently (50% inhibitory concentration (IC50) of 15.34 to 20.89 µM in the Vero cells and RAW 264.7 cells, respectively). Viral RNA and proteins were reduced remarkably. Additionally, TM interfered in the early and late stages of the CHIKV life cycle with efficacy during pretreatment and posttreatment. Moreover, the agonist of the AT1 receptor and an antagonist of PPAR-γ increased CHIKV infection, suggesting that the antiviral potential of TM occurs through modulating host factors. In addition, reduced activation of all major mitogen-activated protein kinases (MAPKs), NF-κB (p65), and cytokines by TM occurred through the inflammatory axis and supported the fact that the anti-CHIKV efficacy of TM is partly mediated through the AT1/PPAR-γ/MAPKs pathways. Interestingly, at a human equivalent dose, TM abrogated CHIKV infection and inflammation significantly, leading to reduced clinical scores and complete survival of C57BL/6 mice. Additionally, TM reduced infection in hPBMC-derived monocyte-macrophage populations in vitro. Hence, TM was found to reduce CHIKV infection by targeting both viral and host factors. Considering its safety and in vivo efficacy, it can be a suitable candidate in the future for repurposing against CHIKV.

Comparison of Clinical Presentation and Vaccine Effectiveness Among Omicron and Non-omicron SARS Coronavirus-2 Patients.

Introduction The rapidly mutating Omicron SARS-CoV-2 variant has replaced the previous dominant SARS-CoV-2 variants like alpha, and delta resulting in the amplification of coronavirus disease 2019 (COVID-19) cases. The present study was conducted to compare the clinical profile and vaccination status in patients infected with Omicron and non-Omicron SARS-CoV-2 variants. Methods All patients who tested positive for coronavirus disease 2019 (COVID-19) during the study period (January 2022 to February 2022) were further tested for detection of SARS-CoV-2 Omicron variant by using Omisure kit (TATA MD CHECK RT-PCR, TATA MEDICAL AND DIAGNOSTICS LIMITED, Tamil Nadu, INDIA). Clinico-demographic factors and vaccination status were compared between both Omicron and non-Omicron groups. Results A total of 1,722 patients who tested positive for COVID-19 were included in the study, of which 656 (38.1%) were Omicron and 1,066 (61.9%) were non-Omicron SARS-CoV-2 variants. Blood group and vaccination status were the major predictors for Omicron. The proportion of male patients was 58.4% in the Omicron group and 57.9% in the non-Omicron group. Maximum cases (86.2%) belonged to >18-60 years age group, 7.3% to >60 years age group, and least to 0-18 years (6.5%). The average age of the study participants was 35.4 ± 14.5 years. Vaccinated participants had less chance of having Omicron than the unvaccinated participants (p-value - 0.003). Fever and loss of smell were found to be significantly associated with the non-Omicron SARS-CoV-2 variant. (p-value < 0.05). Conclusion The present study reflects that the clinical course of the disease is milder in Omicron as compared to the non-Omicron variant. However rapid rise in cases can badly affect the healthcare system demanding good preparedness to tackle all the predicaments. Good Vaccination coverage should be of utmost priority irrespective of the variant type.

MK2a inhibitor CMPD1 abrogates chikungunya virus infection by modulating actin remodeling pathway.

Chikungunya virus (CHIKV) epidemics around the world have created public health concern with the unavailability of effective drugs and vaccines. This emphasizes the need for molecular understanding of host-virus interactions for developing effective targeted antivirals. Microarray analysis was carried out using CHIKV strain (Prototype and Indian) infected Vero cells and two host isozymes, MAPK activated protein kinase 2 (MK2) and MAPK activated protein kinase 3 (MK3) were selected for further analysis. The substrate spectrum of both enzymes is indistinguishable and covers proteins involved in cytokines production, endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodeling and transcriptional regulation. Gene silencing and drug treatment were performed in vitro and in vivo to unravel the role of MK2/MK3 in CHIKV infection. Gene silencing of MK2 and MK3 abrogated around 58% CHIKV progeny release from the host cell and a MK2 activation inhibitor (CMPD1) treatment demonstrated 68% inhibition of viral infection suggesting a major role of MAPKAPKs during late CHIKV infection in vitro. Further, it was observed that the inhibition in viral infection is primarily due to the abrogation of lamellipodium formation through modulation of factors involved in the actin cytoskeleton remodeling pathway. Moreover, CHIKV-infected C57BL/6 mice demonstrated reduction in the viral copy number, lessened disease score and better survivability after CMPD1 treatment. In addition, reduction in expression of key pro-inflammatory mediators such as CXCL13, RAGE, FGF, MMP9 and increase in HGF (a CHIKV infection recovery marker) was observed indicating the effectiveness of the drug against CHIKV. Taken together it can be proposed that MK2 and MK3 are crucial host factors for CHIKV infection and can be considered as important target for developing effective anti-CHIKV strategies.

Inhibition of herpes simplex virus-1 infection by MBZM-N-IBT: in silico and in vitro studies.

INTRODUCTION: The emergence of drug resistance and cross-resistance to existing drugs has warranted the development of new antivirals for Herpes simplex viruses (HSV). Hence, we have designed this study to evaluate the anti-viral activity of 1-[(2-methyl benzimidazole-1-yl) methyl]-2-oxo-indolin-3-ylidene] amino] thiourea (MBZM-N-IBT), against HSV-1. METHOD: Molecular docking was performed to assess the affinity of MBZM-N-IBT for HSV-1 targets. This was validated by plaque assay, estimation of RNA and protein levels as well as time of addition experiments in vitro. RESULT: Molecular docking analysis suggested the inhibitory capacity of MBZM-N-IBT against HSV-1. This was supported by the abrogation of the HSV-1 infectious viral particle formation with the IC50 value of 3.619 µM. Viral mRNA levels were also reduced by 72% and 84% for UL9 and gC respectively. MBZM-N-IBT also reduced the protein synthesis for gC and ICP8 significantly. While mRNA of ICP8 was not significantly affected, its protein synthesis was reduced by 47%. The time of addition experiment revealed the capacity of MBZM-N-IBT to inhibit HSV-1 at early as well as late stages of infection in the Vero cells. Similar effect of MBZM-N-IBT was also noticed in the Raw 264.7 and BHK 21 cells after HSV-1 infection. Supported by the in silico data, this can be attributed to possible interference with multiple HSV targets including the ICP8, ICP27, UL42, UL25, UL15 and gB proteins. CONCLUSION: These results along with the lack of acute oral toxicity and significant anti-inflammatory effects suggest its suitability for further evaluation as a non-nucleoside inhibitor of HSV.

Molecular epidemiology of Japanese encephalitis virus in pig population of Odisha, Assam and Manipur states of India.

Japanese encephalitis virus (JEV) comes under the family Flaviviridae and genus flavivirus. Pigs act as reservoir and amplifying intermediate host for JEV. The current investigation was conducted to understand the prevalence of JEV infection in pigs in three different geographical sites in India (Odisha, Assam and Manipur). Total 857 serum samples were tested by ELISA and RT-PCR, while only RT-PCR was performed in case of 275 tonsils tissues for detection of JEV. It was observed that JEV prevalence was highest in Manipur (positive 39, 25.5% in serum and 10% in tonsil) but lower in Assam (positive 15, 3.8% in serum and 0% in tonsils) and Odisha (positive 7, 1.5% in serum and 3.7% in tonsils). Genotype III (GIII) of JEV was the dominant genotype. Further, analysis of E gene revealed sporadic mutations of S83G, H76P, E78Q, C55S, and S64W along with two consistent mutations V46S and V51I in GIII. Whereas, a single mutation S118N was observed in the GI strain. In conclusion, the high JE virus infection rate of pig in the current locations suggests the need for continuous surveillance of this virus in pigs which will ultimately help to adopt an effective control strategy to prevent the spread of JE infection to human.

Pyrene-appended bipyridine hydrazone ligand as a turn-on sensor for Cu2+ and its bioimaging application.

A pyrene-appended bipyridine hydrazone-based ligand, HL, was synthesized and characterized by spectroscopic methods. Upon complexation with Cu(ii), HL formed a hexanuclear paddlewheel metal-organic macrocycle (MOM) via self-assembly with a high association constant with the molecular formula of [Cu6L6(NO3)6]. Intermolecular and intramolecular π-π interactions were demonstrated in this hexanuclear Cu(ii) complex. Further, it was observed that HL had the potential to detect a trace level of Cu(ii) ion selectively among a wide range of biologically relevant metal ions in aqueous medium at physiological pH. Using HL, it was feasible to sense copper(ii) ions in living cells due to its good cell permeability and high solubility under physiological conditions along with its high IC50 value. The low detection limit, high sensitivity and good reproducibility make this Cu-sensor very promising. The complex (MOM) formed between the ligand and Cu(ii) was found to be 1 : 1 on the basis of fluorescence titrations and was confirmed by ESI-MS. Moreover, single-crystal study of the hexanuclear self-assembled fluorescent species provided better insight into its chemistry, e.g. coordination environment and binding mode, unlike most of the metal sensors due to the lack of a single-crystal structure of the metal sensor complex. Cytotoxicity assay and bioimaging were performed in living cells (Vero cells), giving green fluorescent images. Fluorescence lifetime measurements and theoretical calculations were carried out. The morphology and topographic details on the surface of the metal-organic macrocycle (MOM) were studied by field-emission scanning electron microscopy (FESEM).

The effect of amantadine on an ion channel protein from Chikungunya virus.

Viroporins like influenza A virus M2, hepatitis C virus p7, HIV-1 Vpu and picornavirus 2B associate with host membranes, and create hydrophilic corridors, which are critical for viral entry, replication and egress. The 6K proteins from alphaviruses are conjectured to be viroporins, essential during egress of progeny viruses from host membranes, although the analogue in Chikungunya Virus (CHIKV) remains relatively uncharacterized. Using a combination of electrophysiology, confocal and electron microscopy, and molecular dynamics simulations we show for the first time that CHIKV 6K is an ion channel forming protein that primarily associates with endoplasmic reticulum (ER) membranes. The ion channel activity of 6K can be inhibited by amantadine, an antiviral developed against the M2 protein of Influenza A virus; and CHIKV infection of cultured cells can be effectively inhibited in presence of this drug. Our study provides crucial mechanistic insights into the functionality of 6K during CHIKV-host interaction and suggests that 6K is a potential therapeutic drug target, with amantadine and its derivatives being strong candidates for further development.

P38 and JNK Mitogen-Activated Protein Kinases Interact With Chikungunya Virus Non-structural Protein-2 and Regulate TNF Induction During Viral Infection in Macrophages.

Chikungunya virus (CHIKV), a mosquito-borne Alphavirus, is endemic in different parts of the globe. The host macrophages are identified as the major cellular reservoirs of CHIKV during infection and this virus triggers robust TNF production in the host macrophages, which might be a key mediator of virus induced inflammation. However, the molecular mechanism underneath TNF induction is not understood yet. Accordingly, the Raw264.7 cells, a mouse macrophage cell line, were infected with CHIKV to address the above-mentioned question. It was observed that CHIKV induces both p38 and JNK phosphorylation in macrophages in a time-dependent manner and p-p38 inhibitor, SB203580 is effective in reducing infection even at lower concentration as compared to the p-JNK inhibitor, SP600125. However, inhibition of p-p38 and p-JNK decreased CHIKV induced TNF production in the host macrophages. Moreover, CHIKV induced macrophage derived TNF was found to facilitate TCR driven T cell activation. Additionally, it was noticed that the expressions of key transcription factors involved mainly in antiviral responses (p-IRF3) and TNF production (p-c-jun) were induced significantly in the CHIKV infected macrophages as compared to the corresponding mock cells. Further, it was demonstrated that CHIKV mediated TNF production in the macrophages is dependent on p38 and JNK MAPK pathways linking p-c-jun transcription factor. Interestingly, it was found that CHIKV nsP2 interacts with both p-p38 and p-JNK MAPKs in the macrophages. This observation was supported by the in silico protein-protein docking analysis which illustrates the specific amino acids responsible for the nsP2-MAPKs interactions. A strong polar interaction was predicted between Thr-180 (within the phosphorylation lip) of p38 and Gln-273 of nsP2, whereas, no such polar interaction was predicted for the phosphorylation lip of JNK which indicates the differential roles of p-p38 and p-JNK during CHIKV infection in the host macrophages. In summary, for the first time it has been shown that CHIKV triggers robust TNF production in the host macrophages via both p-p38 and p-JNK/p-c-jun pathways and the interaction of viral protein, nsP2 with these MAPKs during infection. Hence, this information might shed light in rationale-based drug designing strategies toward a possible control measure of CHIKV infection in future.

Chikungunya virus nsP1 interacts directly with nsP2 and modulates its ATPase activity.

Chikungunya virus (CHIKV) is a mosquito-borne virus, which has created an alarming threat in the world due to unavailability of vaccine and antiviral compounds. The CHIKV nsP2 contains ATPase, RTPase, helicase and protease activities, whereas, nsP1 is a viral capping enzyme. In alphaviruses, the four non-structural proteins form the replication complex in the cytoplasm and this study characterizes the interaction between CHIKV nsP1 and nsP2. It was observed that, both the proteins co-localize in the cytoplasm and interact in the CHIKV infected cells by confocal microscopy and immunoprecipitation assay. Further, it was demonstrated through mutational analysis that, the amino acids 1-95 of nsP2 and 170-288 of nsP1 are responsible for their direct interaction. Additionally, it was noticed that, the ATPase activity of nsP2 is enhanced in the presence of nsP1, indicating the functional significance of this interaction. In silico analysis showed close (≤1.7 Å) polar interaction (hydrogen bond) between Glu4, Arg7, 96, 225 of nsP2 with Lys256, 206, Val367 and Phe312 of nsP1 respectively. Hence, this investigation provides molecular characterization of CHIKV nsP1-nsP2 interaction which might be a useful target for rational designing of antiviral drugs.

Regulation of Viral Replication, Apoptosis and Pro-Inflammatory Responses by 17-AAG during Chikungunya Virus Infection in Macrophages.

Chikungunya virus (CHIKV) infection has re-emerged as a major public health concern due to its recent worldwide epidemics and lack of control measures. Although CHIKV is known to infect macrophages, regulation of CHIKV replication, apoptosis and immune responses towards macrophages are not well understood. Accordingly, the Raw264.7 cells, a mouse macrophage cell line, were infected with CHIKV and viral replication as well as new viral progeny release was assessed by flow cytometry and plaque assay, respectively. Moreover, host immune modulation and apoptosis were studied through flow cytometry, Western blot and ELISA. Our current findings suggest that expression of CHIKV proteins were maximum at 8 hpi and the release of new viral progenies were remarkably increased around 12 hpi. The induction of Annexin V binding, cleaved caspase-3, cleaved caspase-9 and cleaved caspase-8 in CHIKV infected macrophages suggests activation of apoptosis through both intrinsic and extrinsic pathways. The pro-inflammatory mediators (TNF and IL-6) MHC-I/II and B7.2 (CD86) were also up-regulated during infection over time. Further, 17-AAG, a potential HSP90 inhibitor, was found to regulate CHIKV infection, apoptosis and pro-inflammatory cytokine/chemokine productions of host macrophages significantly. Hence, the present findings might bring new insight into the therapeutic implication in CHIKV disease biology.

Inhibition of Chikungunya Virus Replication by 1-[(2-Methylbenzimidazol-1-yl) Methyl]-2-Oxo-Indolin-3-ylidene] Amino] Thiourea(MBZM-N-IBT).

Chikungunya virus (CHIKV) infection is one of the most challenging human Arboviral infections with global significance and without any specific antiviral. In this investigation, 1-[(2-methylbenzimidazol-1-yl) methyl]-2-oxo-indolin-3-ylidene] amino] thiourea (MBZM-N-IBT) was synthesised as a molecular hybrid of 2-methyl benzimidazole and isatin-ß-thiosemicarbazone and its anti-CHIKV property was evaluated. The release of infectious virus particles was calculated by plaque assay, expression profile of viral RNA was estimated by RT-PCR and viral protein profiles were assessed by Western blot and FACS analyses. The safety index of MBZM-N-IBT was found to be >21. The CHIKV infectious viral particle formation was abrogated around 76.02% by MBZM-N-IBT during infection in mammalian system and the viral RNA synthesis was reduced by 65.53% and 23.71% for nsP2 and E1 respectively. Surprisingly, the viral protein levels were reduced by 97% for both nsP2 and E2. In the time-of-addition experiment it abrogated viral infection at early as well as late phase of viral life cycle, which indicates about multiple mechanisms for its anti-CHIKV action. In silico analysis justified development of MBZM-N-IBT with good affinities for potential target proteins of CHIKV and related virus. With predictions of good drug-likeness property, it shows potential of a drug candidate which needs further experimental validation.

High rates of co-infection of Dengue and Chikungunya virus in Odisha and Maharashtra, India during 2013.

Dengue viral (DENV) infection is endemic in different parts of India and because of similar primary signs and symptoms, Chikungunya virus (CHIKV) is mostly undiagnosed. Hence, we investigated 204 suspected Dengue cases in a hospital based cross-sectional study in Odisha, India in 2013. It was observed that 50 samples were positive for DENV only, 28 were positive for CHIKV only and interestingly, 28 patients were co-infected with both DENV and CHIKV. Additionally, a total of 18 confirmed Dengue samples from Maharashtra, India were screened for CHIKV and out of those, 15 were co-infected. All CHIKV strains were of East Central South African (ECSA) type and serotype 2 (genotype IV) was predominant in the DENV samples. Additionally, Dengue serotype 1 and 3 were also detected during this time. Further, sequence analysis of E1 gene of CHIKV strains revealed that two substitution mutations (M269V and D284E) were observed in almost 50% strains and they were from co-infected patients. Similarly, sequence analysis of C-prM gene showed the presence of five substitution mutations, (G70S, L72F, N90S, S93N and I150L) in all serotype 1 and two consistent mutations (A101V and V112A) in serotype 2 Dengue samples. Together, it appears that a significantly high number of dengue patients (43, 44.8%) were co-infected with DENV and CHIKV during this study. This emphasizes the need of a routine diagnosis of CHIKV along with DENV for febrile patients. This will be useful in early and proper recognition of infecting pathogen to study the correlation of clinical symptoms with single or co-infection which will ultimately help to implement proper patient care in future.